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Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in DrosophilaSze Ham Chan1., Amy Marie Yu1., Mitch McVey1,2*
1 Department of Biology, Tufts University, Medford, Massachusetts, United States of America, 2 Program in Genetics, Tufts Sackler School of Graduate Biomedical Sciences,
Boston, Massachusetts, United States of America
Abstract
DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or ‘‘alternative’’ end joining, which does not. Alternative end joininghas been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized.Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene andpreviously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative endjoining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking thebreak site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol thetapromotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertionevents. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potentialmechanistic link between alternative end joining and interstrand crosslink repair.
Citation: Chan SH, Yu AM, McVey M (2010) Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila. PLoSGenet 6(7): e1001005. doi:10.1371/journal.pgen.1001005
Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America
Received September 15, 2009; Accepted May 27, 2010; Published July 1, 2010
Copyright: � 2010 Chan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the NSF (MCB-0643253) and by a New Scholar in Aging award to MM from the Ellison Medical Foundation(www.ellisonfoundation.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mitch.mcvey@tufts.edu
. These authors contributed equally to this work.
Introduction
DNA double-strand breaks (DSBs) and interstrand crosslinks
pose serious threats to cell survival and genome stability. Because
these lesions compromise both strands of the double helix, they
impede DNA replication and transcription and therefore must be
removed in a timely and coordinated manner. Interstrand
crosslink repair has been shown to involve a DSB intermediate
in some cases (reviewed in [1]). Therefore, there may be
substantial mechanistic overlap in the processes used during
repair of these two lesions.
Error-free repair of DSBs can be accomplished through
homologous recombination (HR) with an undamaged homologous
template (reviewed in [2]). However, in contexts where suitable
templates for HR do not exist, error-prone repair mechanisms are
also used. For example, non-homologous end joining (NHEJ)
frequently creates small insertions and deletions during DSB
repair, particularly in cases where the broken ends cannot be
readily ligated (reviewed in [3]). Analogously, the use of translesion
DNA polymerases during interstrand crosslink repair can result in
mutations, due to the reduced fidelity of these polymerases [4,5].
Accumulating evidence suggests that NHEJ is composed of at
least two genetically distinct mechanisms. Classical NHEJ (C-
NHEJ) involves the sequential recruitment of two highly conserved
core complexes (reviewed in [6]). First, the Ku70/80 heterodimer
recognizes and binds to DNA ends in a sequence-independent
manner, thereby protecting them from degradation. In many
eukaryotes, Ku70/80 also recruits DNA-PKcs, forming a synaptic
complex that can recruit additional processing enzymes such as
the Artemis nuclease and the X family DNA polymerases mu and
lambda. These proteins expand the spectrum of broken ends that
can be rejoined. The second core complex, composed of DNA
ligase 4, XRCC4, and XLF/Cernunnos, catalyzes ligation of the
processed ends. Depending on the substrate, C-NHEJ can result in
perfect repair of broken DNA, or it can result in small deletions of
1–10 nucleotides and/or insertions of 1–3 nucleotides [7].
Although C-NHEJ can repair blunt-ended substrates, a subset of
C-NHEJ products appear to involve annealing at 1–4 nucleotide
microhomologous sequences on either side of the break.
Alternative end-joining (alt-EJ) is defined as end-joining repair
that is observed in cells or organisms lacking one or more C-NHEJ
components (reviewed in [8]). Alt-EJ in yeast is associated with
deletions larger than those typically created by C-NHEJ, together
with an increased tendency to repair by annealing at micro-
homologous sequences. Ku and ligase 4-independent end joining
observed in mammalian cells also displays an increased tendency
towards use of short microhomologies compared to C-NHEJ
[9,10]. Therefore, alt-EJ is sometimes called microhomology-
mediated end joining (MMEJ) [11]. However, the relationship
between MMEJ and alt-EJ is still unclear, and alt-EJ may comprise
one or more C-NHEJ-independent repair mechanisms [8].
The importance of alt-EJ repair is highlighted by multiple
studies that suggest it may promote chromosome instability and
carcinogenesis. Alt-EJ produces chromosome translocations in
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mouse embryonic stem cells lacking Ku70 [12] and the use of alt-
EJ during V(D)J recombination in C-NHEJ-deficient murine
lymphocytes causes complex chromosome translocations and
progenitor B cell lymphomas [13]. Furthermore, alt-EJ has been
implicated in various translocations associated with chronic
myeloid leukemia and human bladder cancer [14,15]. Important-
ly, alt-EJ also operates during V(D)J rejoining in C-NHEJ-
proficient B lymphocytes [16], suggesting that its role in DSB
repair is not limited to situations where C-NHEJ is defective.
However, alt-EJ is frequently masked by more dominant repair
processes that are essential for vertebrate development, making it
difficult to study. Therefore, its molecular mechanisms and the
proteins involved remain largely unknown.
Several lines of evidence demonstrate that Drosophila is an
excellent model system in which to study alt-EJ in a metazoan. The
Drosophila genome lacks several mammalian C-NHEJ components,
including DNA-PKcs and Artemis. This may predispose flies towards
non-C-NHEJ repair. Consistent with this, we have previously shown
that a DSB caused by excision of a P element transposon in flies is
readily repaired by a DNA ligase 4-independent end-joining process
[17]. Interestingly, although Drosophila orthologs for the Pol X
family DNA polymerases mu and lambda have not been identified
[18], we and others have found evidence for polymerase activity in
Drosophila end-joining repair [17,19,20]. Specifically, end joining in
flies is often associated with the insertion of nucleotides at repair
junctions, frequently involving imperfect repeats of 5–8 nucleotides.
Full or partial templates for the insertions, occasionally possessing
mismatches, can often be identified in adjacent sequences, suggesting
the action of an error-prone polymerase. Similar templated
nucleotides (T-nucleotides) have previously been identified at
translocation breakpoints in human lymphomas [21–23]. Therefore,
T-nucleotides could represent a signature of alt-EJ and may be
informative regarding its molecular mechanisms.
Additional insight into alt-EJ is provided by recent reports
suggesting a mechanistic link between alt-EJ and interstrand
crosslink repair. For example, a study of two Chinese hamster
ovary cell lines sensitive to the crosslinking agent mitomycin C
found that they were also deficient in alt-EJ [24]. Furthermore,
certain interstrand crosslink-sensitive cell lines from Fanconi
Anemia patients are also impaired in DNA-PKcs-independent
rejoining of linearized plasmids [25]. Based on these reports, we
hypothesized that additional mechanistic insight into both
interstrand crosslink repair and alt-EJ could be gained by
searching for mutants defective in both processes. To test this,
we have screened Drosophila mutants that are sensitive to DNA
crosslinking agents for additional defects in alt-EJ repair. In this
work, we describe our studies with one such mutant, mus308.
The mus308 (mutagen sensitive 308) mutant was originally
identified by its extreme sensitivity to interstrand crosslinking
agents but normal resistance to alkylating agents [26]. Subse-
quently, mus308 was found to code for DNA polymerase theta,
which is most similar to A family DNA polymerases such as
Escherichia coli Pol I [27]. Orthologs of polymerase theta (hereafter
referred to as pol h) are found in many metazoans, including
Caenorhabditis elegans, Arabidopsis thaliana, and Homo sapiens, but not in
unicellular eukaryotes, including the yeasts [28–30]. Several lines
of evidence suggest that pol h may play an important role in
maintaining genome stability. Similar to flies, C. elegans with
mutations in POLQ-1 are defective in repair of interstrand
crosslinks [28]. Mice lacking pol h (chaos1 mutants) have a high
frequency of spontaneous and mitomycin C-induced micronuclei
in erythrocytes, consistent with genomic instability [31]. In
addition, vertebrate pol h orthologs have been implicated in a
wide range of repair processes, including base excision repair,
bypass of abasic sites, and somatic hypermutation of immuno-
globulin genes [32–36]. Finally, upregulation of pol h is observed
in a variety of human tumors and is associated with a poor clinical
outcome, suggesting that its overexpression may contribute to
cancer progression [37].
Pol h is unusual in possessing an N-terminal helicase-like domain
and a C-terminal polymerase domain. Although pol h purified from
human cell lines and Drosophila has error-prone polymerase activity
and single-stranded DNA-dependent ATPase activity, helicase
activity has not been demonstrated in vitro [30,38,39]. Therefore, it
remains unclear exactly how the structure of pol h relates to its
multiple functions in DNA repair in different organisms.
We report here that in addition to its role in DNA interstrand
crosslink repair, Drosophila pol h is involved in end-joining repair
of DSBs. This alt-EJ mechanism operates independently of both
Rad51-mediated HR and ligase 4-dependent C-NHEJ. Genetic
analysis using separation-of-function alleles provides support for
distinct roles of both the N- and C-terminal domains of pol h in
alt-EJ. Collectively, our data support a model in which helicase
and polymerase activities of Drosophila pol h cooperate to
generate single-stranded microhomologous sequences that are
utilized during end alignment in alt-EJ.
Results
Drosophila pol h is important for both interstrandcrosslink repair and end-joining repair of DNA double-strand breaks
Drosophila mus308 mutants were initially identified based on
their sensitivity to low doses of chemicals that induce DNA
interstrand crosslinks [26]. To confirm this phenotype, we
assembled a collection of previously identified mus308 mutant
alleles [40,41] and measured the ability of hemizygous mutant
larvae to survive exposure to the crosslinking agent mechloreth-
amine (nitrogen mustard). Of the mutants that we tested, four were
unable to survive exposure to 0.005% mechlorethamine: D2, D5,
2003, and 3294 (data not shown), consistent with their inability to
repair interstrand crosslinks.
Author Summary
DNA double-strand breaks, in which both strands of theDNA double helix are cut, must be recognized andaccurately repaired in order to promote cell survival andprevent the accumulation of mutations. However, error-prone repair occasionally occurs, even when accuraterepair is possible. We have investigated the geneticrequirements of an error-prone break-repair mechanismcalled alternative end joining. We have previously shownthat alternative end joining is frequently used in the fruitfly, Drosophila melanogaster. Here, we demonstrate that afruit fly protein named DNA polymerase theta is a keyplayer in this inaccurate repair mechanism. Geneticanalysis suggests that polymerase theta may be importantfor two processes associated with alternative end joining:(1) annealing at short, complementary DNA sequences,and (2) DNA synthesis that creates small insertions atbreak-repair sites. In the absence of polymerase theta, abackup repair mechanism that frequently results in largechromosome deletions is revealed. Because DNA polymer-ase theta is highly expressed in many types of humancancers, our findings lay the groundwork for furtherinvestigations into how polymerase theta is involved inrepair processes that may promote the development ofcancer.
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To determine the molecular lesions responsible for mechlor-
ethamine sensitivity, we sequenced the entire mus308 coding
region of flies hemizygous for each mutant allele. Pol h possesses
both a conserved N-terminal helicase-like domain and a C-
terminal pol I-like polymerase domain (Figure 1, Figure S1) [30].
Three of the four alleles contain unique sequence changes that are
predicted to affect pol h primary structure (Figure 1, Figure S2,
and Figure S3). The 2003 allele is a nonsense mutation upstream
of the polymerase domain, while the D5 allele is a missense
mutation that alters a highly conserved proline in the conserved N-
terminus. The 3294 allele changes an invariant glycine in the
helicase domain to serine. Interestingly, this residue is conserved in
the related mus301 helicase, but not in other DNA helicases (data
not shown). No mutations were found in the coding sequence of
the D2 allele. Because homozygous D2 flies have undetectable
levels of pol h protein [38], the D2 mutation may affect a
regulatory region of mus308.
One explanation for the extreme sensitivity of mus308 mutants
to mechlorethamine could be a defect in the repair of certain types
of DSB intermediates that are created during crosslink repair. To
test this, we exposed flies hemizygous for each mutant allele to
increasing doses of ionizing radiation (IR). Although IR creates
many different types of lesions, unrepaired DSBs are thought to be
the main cause of cell death following irradiation. All four mus308
mutants survived IR exposures as high as 4000 rads (Figure 2A),
although bristle and wing defects characteristic of apoptotic cell
death were frequently observed at high doses. Drosophila lig4
mutants, which are completely defective in C-NHEJ, also survive
IR doses in excess of 4000 rads [17]. However, spn-A mutants,
which lack the Rad51 recombinase required for strand invasion
during homologous recombination initiation [42], are highly
sensitive to IR [17]. Thus, in Drosophila, HR is the dominant
mechanism used to repair IR-induced DSBs.
To test whether pol h acts to repair IR damage in the absence of
HR, we created mus308 spn-A double mutants and exposed them
to doses of 125–1000 rads. Strikingly, doses as low as 125 rads
resulted in almost complete killing of mus308 spn-A mutants
(Figure 2B). In contrast, lig4 spn-A double mutants are only slightly
more sensitive than spn-A single mutants to IR [17]. Thus, in the
absence of HR, pol h participates in a process crucial for repair of
damage caused by ionizing radiation.
Because interstrand crosslink repair and alternative end joining
have been shown to have partially overlapping genetic require-
ments in mammals [24,25], we hypothesized that the extreme
sensitivity of mus308 spn-A mutants to IR might relate to a role of
pol h in an alternative end-joining mechanism. To explore this
hypothesis, we tested each mus308 mutant allele using a site-
specific double-strand break repair assay that can distinguish
between synthesis-dependent strand annealing (SDSA, a specific
type of HR) and end joining (EJ) (Figure 3A) [43]. We have
previously shown that the majority of end joining observed in this
assay occurs independently of DNA ligase 4, and is therefore a
form of alt-EJ [17]. In this system, excision of a P element (P{wa})
located on the X chromosome is catalyzed by P transposase,
resulting in a 14-kilobase gap relative to an undamaged sister
chromatid. The DNA ends remaining after excision each have 17-
nucleotide non-complementary 39 single-stranded overhangs [44].
These ends are highly recombinogenic and repair by SDSA is
initially favored. However, because repair synthesis in this system
is not highly processive, most repair products that are recovered
from wild-type flies result from incomplete repair synthesis from
one or both sides of the break, followed by end joining of the
nascent DNA (SDSA+EJ events) [45]. To quantitate the
percentage of repair events that derive from each mechanism,
repair events are recovered from male pre-meiotic germline cells
by mating individual males to females homozygous for the P{wa}
element. Each of the resulting female progeny represents a single
repair event that can be classified by eye color. Red eyed-females
inherit a repair event involving homology-dependent synthesis that
generated complementary single-stranded regions that subse-
quently anneal (repair by SDSA). Yellow-eyed females inherit a
chromosome that was repaired by EJ or SDSA+EJ mechanisms
(these repair events are hereafter referred to as (SDSA)+EJ; for
further details, see Materials and Methods).
Overall, the results from the P{wa} assay indicated that mus308
mutants are defective in end-joining repair of DSBs. We observed
no decrease in the percentage of red-eyed progeny recovered from
mus308 mutant males (Figure 3B), suggesting that SDSA repair is
not impaired when pol h is missing or defective. In contrast, all
four mus308 mutant alleles resulted in a significantly decreased
percentage of yellow-eyed progeny relative to wild type (p,0.001,
Kruskal-Wallis test). Because yellow-eyed progeny can only result
from a repair mechanism involving end joining, these data suggest
that pol h is involved in an end-joining process.
To further demonstrate that pol h is not involved in DNA
synthesis during SDSA, we recovered independent (SDSA)+EJ
events in males, isolated genomic DNA, and used PCR to estimate
the approximate amount of DNA repair synthesis that occurred
prior to end joining. The amount of repair synthesis in (SDSA)+EJ
repair products did not differ significantly between wild-type and
mus308 mutant flies (Figure 3C). We conclude that pol h is not
required for DNA synthesis during SDSA, but plays an important
role in end-joining repair following aborted SDSA.
DSB repair in mus308 mutants frequently results inRad51-independent genomic deletions
Mutations that abolish end joining in flies cause an increased
frequency of genomic deletions during repair of site-specific DSBs
Figure 1. Schematic of the mus308 gene. Exons are represented by boxes. The helicase-like domain (light gray shading) and polymerase domain(dark gray shading) are shown. Locations of the D5, 3294, and 2003 point mutations are indicated with arrows (numbers correspond to amino acidpositions in the protein). Not shown is the D2 mutation, which results in severely reduced levels of pol h.doi:10.1371/journal.pgen.1001005.g001
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[46,47]. To determine whether mutation of mus308 also results in
repair-associated deletions, we took advantage of the fact that
deletions can be easily scored in the P element excision assay.
Because P{wa} is inserted in the essential scalloped (sd) gene, repair
events that delete into sd exons cause a scalloped-wing phenotype
when recovered in heterozygous females and lethality in
hemizygous males [48,49]. We observed a substantial increase in
the percentage of deletion-associated repair events isolated from
mus308 mutant males relative to wild type (Figure 3D). Overall, the
total percentage of end-joining repair events involving deletions
recovered from mus308 mutants was elevated from 3- to 26-fold
over wild type, depending on the mus308 allele tested.
Previously, we observed a similar deletion-prone phenotype in
flies lacking the DmBlm protein, which is involved in repair of
Figure 2. Drosophila pol h is required for repair of IR–induced damage in the absence of Rad51. (A) mus308 mutants are not sensitive toionizing radiation. Female flies heterozygous for the indicated mus308 alleles were mated to Df(3R)Sz29/TM3 males and third instar larval progenywere irradiated with increasing doses of IR. The percent survival of mus308 hemizygous mutants was calculated relative to an unirradiated control.Each dose was repeated twice; the average of the two experiments is shown. (B) spn-A mus308 double mutants are extremely sensitive to ionizingradiation. Heterozygous spn-A057mus308D5 females were mated to heterozygous spn-A093mus308D5 males and third instar larval progeny wereirradiated with increasing doses of IR. The percent survival of spn-A mus308 compound heterozygotes was calculated relative to an unirradiatedcontrol. Each dose was repeated at least twice. Error bars represent standard deviations.doi:10.1371/journal.pgen.1001005.g002
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Figure 3. Pol h is involved in end-joining repair. (A) A P element excision assay monitors DSB repair outcomes. The P{wa} transposon is insertedin a 5.2 kb intron of the essential scalloped (sd) gene. A copia retrotransposon flanked by long terminal repeats (LTR) is inserted into an exon of thewhite gene, causing reduced expression of white. Excision of P{wa} results in a 14-kilobase gap relative to an intact sister chromatid. Followingexcision, repair events from the male pre-meiotic germline are recovered in female progeny and the method of repair is inferred from their eye color.SDSA = synthesis-dependent strand annealing, SDSA+EJ = SDSA+end joining, EJ = end joining. Accompanying deletion into sd exons (EJ withdeletion) results in a scalloped-wing phenotype and/or male lethality. (B) Pol h mutants are defective specifically in end-joining repair of DSBs. Eachbar represents the mean percentage of repair events recovered from independent males possessing the indicated mus308 allele in trans to
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DSBs by SDSA [48]. Because our data did not support a role for
pol h in homologous recombination, we expected the deletion-
prone phenotype of mus308 mutants to persist even in SDSA-
deficient flies. To confirm this, we assayed repair following P{wa}
excision in mus308 mutants lacking the Rad51 protein, which
renders them unable to carry out HR repair [42,45].
As expected, PCR analysis of repair products showed that
SDSA was abolished in both spn-A and spn-A mus308 mutants (data
not shown). Approximately 17% of P{wa} chromosomes recovered
from spn-A mutant males showed evidence for end joining at the
17-nucleotide overhangs that are created by P transposase
(Figure 4A and Table 1); the other 83% of P{wa} chromosomes
recovered were presumably uncut. We observed a 30–50%
decrease in end-joining repair products in spn-A mus308 double
mutants compared to spn-A mutants (p,0.001, Kruskal-Wallis
test), confirming a unique role for polh in end joining when HR is
absent. Importantly, mutation of mus308 still caused an increased
percentage of deletions in the absence of Rad51 (Figure 4B). From
these data, we conclude that the deletions formed during break
repair in mus308 mutants are not the result of aborted SDSA.
Rather, they are a consequence of a deletion-prone repair
mechanism that operates in the absence of both SDSA and pol
h-dependent end joining.
During the course of these experiments, we made a number of
observations suggesting that Rad51 and pol h act in parallel and
distinct DSB repair mechanisms. First, we recovered fewer spn-A
mus308 double mutant males than would be predicted from
Mendelian ratios. For example, in crosses between mus308D2 and
mus308D5 heterozygotes, 38% of the progeny were mus308D2/
mus308D5 compound heterozygotes. In contrast, only 16% of
progeny recovered from parallel matings between spn-A057
mus308D2 and spn-A093mus308D5 mutants were spn-A mus308
compound heterozygotes (P,0.05, Fisher’s exact test; Figure 5A).
This difference in viability between mus308 and spn-A mus308
mutants was even more extreme in flies in which excision of P{wa}
was occurring (P,0.01; Figure 5A). In addition, we observed
heightened male sterility in various combinations of spn-A mus308
mutants undergoing P{wa} transposition, with 51% of the double
mutant males unable to produce viable progeny in the most severe
allele combination (Figure 5B). Finally, we observed morpholog-
ical abnormalities, specifically abdominal closure defects and
aberrant cuticle banding patterns, in 100% of spn-A093mus308D5/
spn-A057mus308D2 double mutants (Figure 5C). These defects were
more severe in the double mutants experiencing P{wa} transpo-
sition, but were not apparent in either mus308 or spn-A single
mutants. From these data, we conclude that Rad51 and pol hparticipate in independent pathways required for repair of DSBs
that arise during both endogenous developmental processes and
during P element transposition.
Pol h–mediated end joining is distinct from C-NHEJP element ends are good substrates for DNA ligase 4-independent
end joining [17]. Based on the results presented above, it seemed
likely that pol h is involved in an end-joining process different from
C-NHEJ. To formally test this, we repeated the P{wa} assay in lig4
mus308 double mutants that lack DNA ligase 4 and are unable to
repair DSBs by C-NHEJ. Unlike spn-A mus308 mutants, we
observed no viability, fertility, or morphological defects in lig4
mus308 mutants. We also observed no defect in HR repair in the
double mutants (Figure 4C), consistent with results obtained using
mus308 single mutants. In contrast, we observed a further decrease
in the percentage of end joining repair products recovered from lig4
mus308 double mutants relative to mus308 mutants, from 3.0% to
1.3% (P,0.01, Kruskal-Wallis test). Previously, we have shown that
end-joining repair of DSBs induced by P{wa} excision is unaffected
in lig4 mutants [17]. Therefore, the removal of pol h-mediated end
joining reveals a previously hidden role for DNA ligase 4 in the
repair of DSBs created by P transposase. Strikingly, although only
50% of end-joining products isolated from mus308 mutants involved
large, male-lethal deletions, 100% of end-joining products recov-
ered from lig4 mus308 mutant males were associated with large
deletions (Figure 4D).
From these results, we conclude that at least three distinct
mechanisms for end-joining repair exist in Drosophila. One, which
corresponds to C-NHEJ, requires DNA ligase 4 and other
canonical NHEJ proteins, including XRCC4, Ku70, and Ku80
[46,47,50]. Another mechanism, which is at least partially
independent of DNA ligase 4, is defined by a requirement for
pol h and corresponds to alt-EJ. Interestingly, alt-EJ appears to be
used more frequently than C-NHEJ, at least for the repair of P
element-induced breaks. In the absence of these two repair
processes, a Rad51-independent backup mechanism characterized
by extensive genomic deletions operates at low efficiency.
Pol h has two distinct functions in alternative end joiningAlt-EJ repair in Drosophila is frequently associated with annealing
at microhomologous sequences of more than four nucleotides and
with long DNA insertions at repair junctions [8]. To determine
whether pol h-dependent end joining involves either of these types of
repair, we sequenced repair junctions obtained from spn-A and spn-A
mus308 double mutants following P{wa} excision. Because we
sequenced only one junction per male germline, each junction
analyzed represents an independent repair event. Five distinct
junction types were identified. Three of these types are characteristic
of junctions arising from C-NHEJ in mammalian systems [7]:
junctions involving small, 1–3 base pair insertions, junctions involving
annealing at 1–3 nucleotide microhomologies, and junctions for
which no microhomologies can be identified (apparent blunt end
junctions). The other two types of junctions, characteristic of alt-
EJ [8], involve annealing at 5–10 nucleotide microhomologous
sequences or insertions of more than three base pairs.
Approximately 58% of junctions from spn-A mutants showed
structures considered typical of C-NHEJ repair, while 29%
involved annealing at 5–10 nucleotide microhomologies and
13% had insertions of greater than three base pairs (Figure 6A
and Table 1). Potential templates for the larger insertions could
almost always be identified in flanking sequences. These insertions
may be analogous to T-nucleotides that have been observed at
translocation breakpoint junctions isolated from certain human
cancers [21–23].
Df(3R)Sz29. (SDSA)+EJ = repair involving HR initiation followed by end joining or repair involving only end joining. Number of independent malesassayed: wild type = 101, D2 = 155, D5 = 161, 2003 = 155, 3294 = 100. Error bars represent SEM. (*** indicates p,0.001, Kruskal-Wallis non-parametricANOVA). p .0.5 for all comparisons between different mus308 alleles. (C) Synthesis during HR is not impaired in pol h mutants. Each bar representsthe percentage of repair events recovered from yellow-eyed females that showed evidence for repair synthesis of at least the indicated length.Number of independent repair events: wild type = 32, D2 = 28, D5 = 53. (D) Pol h mutants have an increased frequency of DSB repair events withflanking deletions. For all genotypes, small deletions ,3.6 kilobases upstream or ,1.6 kilobases downstream of P{wa} were detected by PCR. Largedeletions resulted in scalloped-winged female progeny and/or male lethality.doi:10.1371/journal.pgen.1001005.g003
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When we sequenced repair junctions from spn-A mus308
mutants, we observed two distinct patterns, depending on the
mus308 alleles used. For both the D2/2003 and D5/2003 allele
combinations, the percentage of junctions involving annealing at
long microhomologies was significantly decreased (P,0.01,
Fisher’s exact test; Figure 6A, Table 2, and Table 3). Only 12%
of D2/2003 junctions possessed an insertion greater than three
base pairs, compared to 44% of junctions recovered from males
with the D5 and 2003 alleles. In addition, most insertions isolated
from D5/2003 males were highly complex and had multiple copies
of imperfect repeats of T-nucleotides. Similar results were
obtained with the D5/3294 allele combination (data not shown).
An overall comparison of insert length showed that flies with wild-
type mus308 alleles had an average insert length of 5.5 nucleotides,
compared to 3.8 nucleotides for D2/2003 mutants and 13.3
nucleotides for D5/2003 mutants.
Figure 4. Pol h–dependent end joining acts independently of HR and C-NHEJ. (A) Decreased end joining in mus308 mutants does notdepend on homologous recombination. Each bar represents the mean percentage of end-joining events recovered from independent males of eachgenotype. Number of independent males assayed: spn-A = 63; spn-A057mus308D2/spn-A057mus3082003 = 216; spn-A057mus308D2/spn-A057mus308D2 = 33;spn-A093mus308D5/spn-A057mus3082003 = 80; spn-A093mus308D5/spn-A093mus3083294 = 128. Error bars represent SEM. ** indicates p,0.01, *** indicatesP,0.001 (Kruskal-Wallis non-parametric ANOVA). (B) Genomic deletions during DSB repair in mus308 mutants do not depend on initiation of HR. Eachbar represents the percentage of yellow-eyed female progeny with scalloped wings (representing large flanking deletions) recovered from males ofindicated genotype. (C) Mutation of mus308 in a lig4169a background further reduces end-joining repair relative to mus308 single mutants. Each barrepresents the mean percentage of repair events recovered from wild type, mus3082003/Df(3R)kar3l, or lig4169a; mus3082003/Df(3R)kar3l males. Numberof independent males assayed: wild type = 101, mus308 = 182, lig4 mus308 = 57. Error bars represent SEM. ** indicates p,0.01, *** indicates P,0.001(Kruskal-Wallis non-parametric ANOVA). (D) Genomic deletions always occur during DSB repair in lig4 mus308 mutants. All yellow-eyed repair eventsrecovered from lig4169a; mus3082003/Df(3R)kar3l males had large deletions that resulted in male lethality and/or female progeny with scalloped wings.Number of independent repair events assayed: wild type = 101, mus308 = 167, lig4 mus308 = 22.doi:10.1371/journal.pgen.1001005.g004
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In summary, both mus308 mutant combinations significantly
abrogated annealing at long microhomologies during alt-EJ repair.
However, we observed a distinct difference in repair junctions
recovered from males harboring the D2 allele, which greatly
reduces overall pol h protein levels [38], compared to flies with the
D5 allele, which alters a conserved residue near the helicase-like
domain. These results suggest that pol h has two distinct functions
in alt-EJ: one that promotes the annealing of long microhomo-
logous sequences during end alignment, and another that is
responsible for complex T-nucleotide insertions. Flies with the D2
allele are impaired in their ability to carry out both the annealing
and insertion functions, whereas flies possessing the D5 separation-
of-function allele cannot perform the microhomology annealing
function but can still produce complex insertions.
Pol h operates in alternative end joining ofcomplementary-ended double-strand breaks
P element-induced breaks are unique in that they possess 17-
nucleotide non-complementary ends that are poor substrates for
Table 1. P{wa} repair junctions recovered from spn-A093/057 mutants.
Type of repair event Sequence 59 of breakaMicrohomology/insertedsequence Sequence 39 of breaka Number of isolates
Original sequence acccagacCATGATGAAATAACATA - TATGTTATTTCATCATGacccagac -
Long microhomologyb
acccagac (CATgATGA)c cccagac 14
acccagac (CATGA) cccagac 4
noned (TGACCCAGAC) - 2
Short microhomology
acccagacCATG (ATG) TTATTTCATCATGacccagac 1
acccagacCA (TGA) cccagac 1
acccagacCATGATGAAATAA (CAT) Gacccagac 3
acccagacCATGATGAAATAAC (AT) GTTATTTCATCATGacccagac 14
acccagacCATGATGAAATAACA (TA) TGTTATTTCATCATGacccagac 5
acccagacCATGATGAAA (TA) TGTTATTTCATCATGacccagac 1
acccagacCATGATGAA (AT) GTTATTTCATCATGacccagac 1
noned (T) TCATGacccagac 1
Blunt join
acccagacCATGATGAAATA - TTATTTCATCATGacccagac 1
Insertione
acccagacCATGATGAAATAACATA A CATCATGacccagac 1
acccagacCATGATGA G CATCATGacccagac 1
acccagacCATGATGAAATAACATA AC ATGTTATTTCATCATGacccagac 3
acccagacCATGATGAAATAACAT GT TATGTTATTTCATCATGacccagac 1
acccagacCATGATGA AC ATGTTATTTCATCATGacccagac 1
acccagacCA AG ATGacccagac 1
acccagacCATGATGAAATAACATA TTC ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT GTTA TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TTTAT TGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT TTATCA TGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT TTAACATAAC ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TTATTATTATA TTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT GAAATAATAAC ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT GTATTACATAAC ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAA TAATAATAATATAA TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATA TCATGAAATATCATA TCATCATGacccagac 1
a Uppercase letters represent the 17-nucleotide 39 single-stranded tails that remain following transposase action, lowercase letters correspond to the 8 base pair targetsequence duplicated upon P element insertion.b Microhomologies (in parentheses) are sequences that could have been derived from either side of the break site. Long microhomologies were five or morenucleotides, while short microhomologies were three or fewer nucleotides.c represents an 8-nucleotide imperfect microhomology.d indicates a deletion that extends past the 8 base pair target sequence.e Insertions were identified as any sequence not present at the original break site. Templated insertions and corresponding potential templates in flanking sequencesare underlined.doi:10.1371/journal.pgen.1001005.t001
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C-NHEJ. To test whether the results obtained with P elements can
be generalized to other types of breaks, we used the I-SceI
endonuclease and the previously characterized [Iw]7 reporter
construct [50] to create site-specific DSBs in wild-type flies and
flies lacking either DNA ligase 4 or pol h. I-SceI produces a DSB
with 4-nucleotide complementary overhangs that can be directly
ligated through a C-NHEJ mechanism [50,51]. Accurate repair
regenerates the original I-SceI recognition sequence, which can
then be cut again, while inaccurate end-joining repair abolishes
further cutting. We utilized an hsp70 or ubiquitin-driven I-SceI
construct integrated on chromosome 2 to drive high levels of I-SceI
expression [50,52]. Nearly 100% of repair events that we
recovered involved gene conversion (HR repair from the
homologous chromosome) or inaccurate end-joining (data not
shown). In the [Iw]7 system, both gene conversion events and large
deletions that remove the white marker gene are phenotypically
indistinguishable. PCR analysis confirmed that many repair events
recovered from mus308 mutants involved large deletions (.700
base pairs, data not shown). Our subsequent analysis focused on
the characterization of repair events involving smaller deletions.
Twenty-three percent of I-SceI repair junctions isolated from
wild-type flies possessed insertions of more than 3 base pairs
(Figure 6B). This percentage was significantly increased to 46% in
lig4 mutants (P,0.01, Fisher’s exact test), consistent with increased
use of alt-EJ in the absence of C-NHEJ. If pol h plays a general
role in insertional mutagenesis during alt-EJ repair, one would
predict that the frequency and length of insertions following I-SceI
cutting should decrease in mus308 mutants. Indeed, the percentage
of large insertions decreased to 9% in mus308 mutants (P = 0.03,
Fisher’s exact test). Wild-type flies had an average insertion length
of 7.6 base pairs, compared to 4.2 base pairs for mus308 mutants.
Strikingly, no mus308 insertion was longer than twelve base pairs,
while insertions of more than twenty base pairs occurred in both
wild type and lig4 mutants. Because microhomologies of greater
than four base pairs are not present near the I-SceI cut site in this
construct, repair involving annealing at long microhomologies was
not observed.
Surprisingly, the total percentage of repair junctions with short,
1–3 base pair insertions was not decreased in lig4 mutants relative
to wild type (17% vs. 13%, respectively). Furthermore, the
percentage of junctions involving annealing at 1–3 nucleotide
microhomologies was also similar between the two genotypes (25%
for lig4 mutants vs. 34% for wild type). These two types of
junctions have historically been associated with ligase 4-dependent
C-NHEJ repair. Our results suggest that this may not be the case.
Indeed, a fine-level sequence analysis of I-SceI repair junctions that
we have recently conducted suggests that alt-EJ may produce C-
NHEJ-like junctions in certain sequence contexts [53]. Neverthe-
less, our data obtained using two independent site-specific DSB
repair assays strongly suggest that C-NHEJ and alt-EJ represent at
least partially independent mechanisms for the repair of DSBs and
that pol h plays an important role in the generation of T-
nucleotide insertions during alt-EJ repair of both P element and I-
SceI-induced breaks.
Figure 5. Flies lacking Rad51 and Pol h have synergisticphenotypic defects. (A) spn-A mus308 mutants are sub-viable. Femalesand males with the P{wa} transposon and heterozygous for the spn-A057
or spn-A093 and mus308D2 or mus308D5 alleles were interbred and thepercentage of viable compound heterozygous progeny was determined.The crosses were repeated in the presence of P transposase. Each barrepresents three independent experiments with at least 300 progeny perexperiment. Error bars represent SEM. * indicates p,0.05, ** indicatesP,0.01 (unpaired T test). (B) Increased male sterility in spn-A mus308double mutant males. Each bar represents the percentage of spn-A or
spn-A mus308 males that produced no progeny. Number of independentmales assayed: spn-A = 57; spn-A057mus308D2/spn-A057mus3082003 = 305;spn-A057mus308D2/spn-A057mus308D2 = 50; spn-A093mus308D5/spn-A057
mus3082003 = 102; spn-A093mus308D5/spn-A093mus3083294 = 100. (C) Muta-tion of both spn-A and mus308 results in external morphological defectssuch as abdominal cuticle mispatterning (note the severe disruption ofnormal segmental banding patterns). Pictured are representative wildtype and spn-A057mus308D2/spn-A057mus3082003 males in which Pelement excision is occurring.doi:10.1371/journal.pgen.1001005.g005
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Discussion
Several studies have identified proteins important for end-
joining repair of DSBs in the absence of C-NHEJ factors in yeasts
[11,54,55]. More recently, the Mre11 protein has been identified
as an important alt-EJ component in vertebrate systems [10,56–
59]. However, much remains uncertain about the genetics or
mechanisms of alt-EJ in metazoans. Our previous work demon-
strated that end-joining repair of P element-induced breaks can
occur independently of DNA ligase 4, suggesting the presence of a
highly active alternative end-joining mechanism in Drosophila
[17]. We have now identified the mus308 gene, encoding DNA
polymerase theta, as a critical component of alt-EJ. Pol h-
dependent alt-EJ operates in parallel to C-NHEJ to promote
repair of both P element and I-SceI-induced breaks. Because we
observed similar alt-EJ defects with four different mus308 mutant
alleles, several of which were studied in trans to at least two
independently-derived deficiencies, we consider it highly unlikely
that the phenotypes we observed are due to second-site mutations
or other differences in genetic background.
Importantly, pol h does not appear to participate in homology-
directed repair. HR repair of DSBs following P{wa} excision is
thought to proceed largely through synthesis-dependent strand
annealing (SDSA) [60]. We observed that SDSA frequencies in the
P{wa} assay were similar in wild type and mus308 mutants.
Although we did not formally test for a role of pol h in single-
strand annealing (SSA), a non-conservative HR pathway that
involves annealing at direct repeats larger than 25 nucleotides, a
separate study demonstrated that pol h has no effect on SSA repair
[47]. In addition, comparison of the DNA sequences located 3
kilobases to either side of P{wa} by BLAST does not reveal any
significant similarities of more than 20 nucleotides. Therefore, it
seems unlikely that the repair observed in mus308 mutants arose
through an SSA mechanism.
Three findings suggest that mus308-dependent alt-EJ is an
important repair option for both cell and organism survival in flies,
particularly in the absence of homologous recombination. First,
spn-A mus308 double mutants are sub-viable and have severe
defects in adult abdominal cuticle closure, consistent with a high
level of apoptosis in rapidly proliferating histoblasts during
pupariation. Second, spn-A mus308 double mutant males under-
going P{wa} excision have up to 30-fold increased sterility relative
to spn-A mutants. Third, mus308 mutant males undergoing I-SceI
cutting show premature sterility and produce few progeny. The
few germline repair events that are recovered from each male are
frequently clonal, suggesting extensive germline apoptosis (A. Yu,
unpublished data).
Evidence for two functions of pol h in alternative endjoining
Pol h orthologs characterized from a variety of metazoans
possess both helicase-like and DNA polymerase domains
Figure 6. Pol h is required for two distinct classes of alt–EJ repairproducts. (A) Annealing at long microhomologies is greatly decreased inpol h mutants. Repair junctions isolated following P{wa} excision wereamplified from spn-A and spn-A mus308 females of the indicatedgenotypes and sequences were aligned to the original P{wa} chromo-some. White, blunt joins; hatched, 5–10 bp microhomologies; light gray,1–4 bp microhomologies; dark gray, 1–3 bp insertions; black, $4 bpinsertions. Number of junctions sequenced: spn-A = 68, spn-A093mus308D5/spn-A057mus3082003 = 23, spn-A057mus308D2/spn-A057mus3082003 = 25. Thepercentage of 5–10 bp microhomologies was significantly decreased in
both spn-A mus308 mutants (P,0.01, Fisher’s exact test). (B) Reducedfrequency of $4 bp insertions during inaccurate end-joining repair of achromosomal I-SceI DSB in mus308 mutants. Complementary ended DSBswere induced by expression of I-SceI endonuclease in the pre-meioticgermline of wild type (n = 70), lig4169a (n = 83), and mus3082003/Df(3R)kar-Sz29 (n = 57) males, and independent inaccurate end-joining repairjunctions were sequenced. White, blunt joins; light gray, 1–4 bpmicrohomologies; dark gray, 1–3 bp insertions; black, $4 bp insertions.No .4 bp microhomologies are available near the DSB in this system.* indicates P = 0.03, ** indicates P,0.01, relative to wild type (Fisher’sexact test).doi:10.1371/journal.pgen.1001005.g006
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[27–31,35]. Pol h purified from both Drosophila and human cells
has a Pol I-like polymerase activity and single-stranded DNA-
dependent ATPase activity [30,38]. However, DNA helicase
activity of the purified protein remains to be demonstrated.
Although our experiments did not formally test for helicase activity
of pol h, our results are consistent with pol h having a DNA
unwinding or strand displacement function. Flies with the D5 and
3294 mutations (located in or near the conserved helicase domain)
produce repair products with complex T-nucleotide insertions but
not products involving annealing at long microhomologies. The
D5 and 3294 alleles may therefore encode proteins that retain
polymerase activity but lack unwinding activity, resulting in an
inability to expose internal microhomologous sequences. Because
the microhomologies used in repair following P element excision
are often located in the 17-nucleotide 39 single-stranded tails, pol hmay also be important for the unwinding of secondary structures
that form in single-stranded DNA. Alternatively, the DNA-
dependent ATPase activity demonstrated by pol h might represent
an annealing function of the protein that is required during alt-EJ.
Such an annealing activity was recently described for the human
HARP protein, which is able to displace stably bound replication
protein A and rewind single-stranded DNA bubbles [61].
One notable aspect of alt-EJ in Drosophila is the large
percentage of repair junctions with templated insertions. These
insertions may be ‘‘synthesis footprints’’ that are formed during
the cell’s attempt to create microhomologous sequences that can
be used during the annealing stage of alt-EJ when suitable
endogenous microhomologies are not present or are not long
enough to allow for stable end alignment. Indeed, analysis of the
insertions from I-SceI repair junctions suggests a model involving
local unwinding of double-stranded DNA and iterative synthesis of
3–8 nucleotide runs [53]. The P{wa} repair junctions isolated from
mus308D2/mus3082003 mutants are consistent with an important
(but not exclusive) role for the polymerase domain of pol h in the
synthesis of T-nucleotides.
We speculate that pol h may be involved in both DNA
unwinding and repair synthesis during alt-EJ (Figure 7). Linking
these two activities in one protein would provide a convenient
mechanism for creating longer microhomologies that could
increase the thermodynamic stability of aligned ends prior to the
action of a DNA ligase. Studies based on the crystal structure of a
dual function NHEJ polymerase-ligase protein found in Mycobac-
terium tuberculosis suggest that a synaptic function for an NHEJ
polymerase is plausible [62]. Because ligase 4 is not involved in alt-
EJ in Drosophila, another ligase must be involved in the ligation
step. Studies from mammalian systems have identified DNA ligase
3 as a likely candidate [63,64].
Potential roles of pol h in DNA interstrand crosslink repairPol h was originally identified in Drosophila based on the
inability of mus308 mutants to survive exposure to chemicals that
induce DNA interstrand crosslinks. A crucial question posed by
our findings is whether pol h performs a common function during
the repair of both DSBs and interstrand crosslinks. The C. elegans
Table 2. P{wa} repair junctions recovered from spn-A093/057 mus308D5/2003 mutants.
Type ofrepair event Sequence 59 of break Microhomology/inserted sequence Sequence 39 of break
Number ofisolates
Original sequence acccagacCATGATGAAATAACATA - TATGTTATTTCATCATGacccagac -
Short microhomologya
acccagacCATGATGAAATAA (CAT) CATGacccagac 2
acccagacCATG (ATG) TTATTTCATCATGacccagac 1
acccagacCATGATGAAATAAC (AT) GTTATTTCATCATGacccagac 5
acccagacCATGATGAAATAACA (TA) TGTTATTTCATCATGacccagac 2
Blunt join
acccagacCATGATGAAATAACATA - TTATTTCATCATGacccagac 1
Insertionb
acccagacCATGATGAAATAACAT G TGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA AC ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT GTTA TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TGTA TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACA GTGAA ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT GTTATGT TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT GTTATACA TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TATGTTATAACA TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAA TCATGTTATTTC ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TAACATGAATAAC ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TATAATGTTATAACATAT-AACATATGTTATGAAATAATAACA
TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT CATCATTTATCATTTATTATTATTA-TTATTTATTATTATTTATTATTTA
TTATTTCATCATGacccagac 1
a Microhomologies (in parentheses) are sequences that could have been derived from either side of the break site.b Insertions were identified as any sequence not present at the original break site. Templated insertions and corresponding potential templates in flanking sequencesare underlined.doi:10.1371/journal.pgen.1001005.t002
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pol h ortholog, POLQ-1, is also required for resistance to
interstrand crosslinks and acts in a pathway that is distinct from
HR but depends on CeBRCA1 [28]. In S. cerevisiae, several repair
mechanisms are utilized during interstrand crosslink repair,
including nucleotide excision repair (NER), HR, and translesion
synthesis [65,66]. Given our results and the findings from C.
elegans, it seems unlikely that the role of pol h in interstrand
crosslink repair involves a function in HR.
In human cells, exposure to agents that induce interstrand
crosslinks causes a shift in repair mechanisms that leads to
increased use of non-conservative pathways associated with
complex insertions and deletions [67]. Furthermore, interstrand
crosslinks can cause frequent recombination between direct
repeats [68,69], suggesting that single-strand annealing may
provide a viable mechanism for interstrand crosslink repair. The
single-strand annealing model of interstrand crosslink repair posits
that NER-independent recognition and processing of the cross-
linked DNA is followed by generation of single-stranded regions
flanking the crosslink and annealing at repeated sequences.
Because alt-EJ frequently proceeds through annealing at short
direct repeats, it is tempting to speculate that the role of pol h in
interstrand crosslink repair might be to expose and/or promote
the annealing of microhomologous single-stranded regions that
flank the crosslinked DNA. Consistent with this model, the initial
incision step made after recognition of the interstrand crosslink
remains normal in mus308 mutants [26]. Alternatively, pol h might
utilize its polymerase activity and nearby flanking sequences as a
template to synthesize short stretches of DNA that could be used to
span a single-stranded gap opposite of a partially excised crosslink.
Such a model has been proposed to explain the formation of
microindels in human cancers [70]. We are currently testing these
two models using helicase- and polymerase-specific mus308 mutant
alleles.
Pol h and alternative end joining: promoting genome(in)stability
Although it seems counterintuitive, alt-EJ likely functions in
some situations to promote genome stability. As evidence of this,
we found that DSB repair following P element excision in mus308
mutant flies frequently results in genomic deletions of multiple
kilobases. A similar deletion-prone phenotype was previously
observed in mus309 mutants, which lack the Drosophila BLM
ortholog [43,71]. Epistasis analysis demonstrated that the mus309
deletion phenotype depends on Rad51, implying that DmBlm acts
after strand invasion during HR and that the deletions observed in
mus309 mutants are likely a result of failed SDSA [48]. In contrast,
the deletions observed in mus308 mutants do not depend on
Rad51, demonstrating that the function of pol h in DSB repair is
independent of HR. The deletion phenotype is exacerbated in lig4
mus308 double mutants, suggesting that C-NHEJ and alt-EJ
represent two parallel mechanisms to prevent deletions. In the
absence of these two end-joining options, resection at the broken
ends may continue unchecked, resulting in extensive genomic
deletions that are generated by an unknown Rad51-independent
repair mechanism. Therefore, both C-NHEJ and alt-EJ function
to prevent overprocessing of broken DNA ends and extreme
degradation of the genome. Microhomology-mediated end
Table 3. P{wa} sequences recovered from spn-A093/057 mus308D2/2003 mutants.
Type of repair event Sequence to left of breakMicrohomology/insertedsequence Sequence to right of break Number of isolates
Original sequence acccagacCATGATGAAATAACATA - TATGTTATTTCATCATGacccagac -
Long microhomologya
acccagac (CATgATGA) cccagac 1
Short microhomology
acccagacCATGATGAAATAAC (AT) GTTATTTCATCATGacccagac 7
acccagacCATGATGAAATAACA (TA) TGTTATTTCATCATGacccagac 3
acccagacCATGATGA (AT) GTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACA (T) TATTTCATCATGacccagac 1
Blunt join
acccagacCATGATGAAATAACATA - TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACAT - TATGTTATTTCATCATGacccagac 1
Insertionb
acccagacCATGATGAAATAACAT T TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA CA ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA AC ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TA TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAA TGT TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TGT TATGTTATTTCATCATGacccagac 2
acccagacCATGATGAAATAACATA ACATAA ATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TATACCG TATGTTATTTCATCATGacccagac 1
acccagacCATGATGAAATAACATA TGTTATAAC ATGTTATTTCATCATGacccagac 1
a Microhomologies (in parentheses) are sequences that could have been derived from either side of the break site.b Insertions were identified as any sequence not present at the original break site. Templated insertions and corresponding potential templates in flanking sequencesare underlined.doi:10.1371/journal.pgen.1001005.t003
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joining, which shares many features with alt-EJ, has been proposed
to perform a similar function in urothelial cells [72].
Nonetheless, alternative end-joining repair can also be genome
destabilizing, as demonstrated by an increasing number of reports
linking it to cancer. We have shown that complex insertions
observed in alternative end-joining products are more frequent in
flies possessing pol h. These insertions, which are often combina-
tions of nucleotides derived from several templates inserted in both
direct and reverse-complement orientations, are remarkably similar
to T-nucleotide insertions found in translocation breakpoints
reconstructed from follicular and mantle cell lymphomas (reviewed
in [73]). Therefore, if pol h also functions in alternative end joining
and T-nucleotide generation in mammals, it might be an important
factor involved in translocation formation.
A recent study suggests that pol h levels are tightly regulated in
humans and that loss of this regulation may promote cancer
progression [37]. The protein is primarily found in lymphoid tissues
but is upregulated in lung, stomach, and colon cancers. Further-
more, high levels of pol h expression correlate with poorer clinical
outcomes. Intriguingly, pol h is regulated by endogenous siRNAs in
Drosophila [74,75], although the significance of this regulation is
currently unclear. We suggest that polh-mediated alt-EJ serves as a
medium-fidelity repair option used by cells when precise repair
cannot be carried out for any number of reasons. As such, it
prevents extreme loss of genetic information. However, its error-
prone nature requires tight regulation, which, when lost, may lead
to excessive inaccurate repair and ultimately, carcinogenesis.
ConclusionsThe results described here establish that Drosophila pol h plays
two distinct roles in an alternative end-joining mechanism operating
in parallel to canonical DNA ligase 4-mediated C-NHEJ. This novel
finding lays the groundwork for future studies focusing on the
specific roles of the pol h helicase-like and polymerase domains in
alt-EJ and DNA interstrand crosslink repair. Whether pol h plays a
similar role in alt-EJ in other organisms, including mammals,
remains to be determined. Regardless, these studies reveal an
unexpected role for DNA polymerase h that is required for genomic
integrity in Drosophila and possibly other metazoans.
Materials and Methods
Drosophila stocks and mus308 allelesAll flies were maintained on standard cornmeal-based agar food
and reared at 25uC. The mus308 D2 and D5 stocks were obtained
from the Bloomington Stock Center and the 2003 and 3294 stocks
were from the Zuker collection [76]. To identify mutations in these
stocks, genomic DNA was isolated from flies harboring each allele
in trans to Df(3R)Exel6166 and PCR was performed with primers
specific to overlapping regions of the entire coding sequence. PCR
products were sequenced and the sequence was compared to the
Drosophila reference sequence release 5.10. Sequence changes
unique to each allele were verified by sequencing in both
orientations. The lig4169a [17], spn-A093 and spn-A057 [42] stocks
harbor null alleles of DNA ligase 4 and Rad51, respectively.
Figure 7. Model for pol h function in alt–EJ. Pol h is drawn as a bipartite protein, with a helicase domain and a polymerase domain separated by aflexible linker region. (A) Initially, the helicase activity of pol h unwinds short stretches of double-stranded DNA to expose pre-existing microhomologoussequences. (B) These microhomologies (MH) are used to align the broken ends to provide a template for pol h polymerase activity. The unwindingactivity could also serve to make the polymerase more processive. (C) Processing of the ends and ligation results in repair accompanied by a deletion. (D)In cases where the ends do not remain stably aligned, annealing at other microhomologies closer to the break site could result in the insertion of T-nucleotides. Multiple rounds of unwinding, synthesis, and alignment could result in the complex insertions that are often observed in alt-EJ in flies.doi:10.1371/journal.pgen.1001005.g007
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Mutagen sensitivity studiesFor mechlorethamine sensitivity assays, balanced, heterozygous
parents were crossed to Df(3R)Exel6166 and allowed to lay eggs in
vials containing 10mL of food for three days, after which they were
moved to new vials for two additional days. The first vials were
treated with 250mL of 0.005% mechlorethamine dissolved in
ddH2O, while the second vials were treated only with ddH2O.
Survival was calculated as the number of homozygous mutant
adults divided by the total number of adults that eclosed within 10
days of treatment. Ratios were normalized to untreated controls
for each set of vials (five to eight sets of vials were counted for each
experiment). For ionizing radiation sensitivity assays, heterozygous
parents laid eggs on grape-juice agar plates for 12 hr. Embryos
developed at 25uC until larvae reached third-instar stage, at which
point they were irradiated in a Gammator 1000 irradiator at a
dose rate of 800 rads/min and larvae were transferred to food-
containing bottles. Relative survival rates were calculated as
above.
P{wa} assayRepair of DNA double-strand breaks was monitored after
excision of the P{wa} transposon as described previously [43,77].
P{wa} was excised in males using a second chromosome transposase
source (CyO, H{w+,D2–3}) and individual repair events were
recovered in female progeny over an intact copy of P{wa}. Females
with two copies of P{wa} have apricot eyes [78]. Progeny with red
eyes possess a repair event involving HR with annealing of the copia
LTRs. A fraction of apricot-eyed females also possess HR repair
events, but these cannot be distinguished from chromosomes in
which no excision event occurred (using the CyO, H{w+,D2–3}
transposase source, ,80% of apricot-eyed female progeny inherit a
non-excised P{wa} element). Yellow-eyed females harbor a repair
event in which repair is completed by end joining.
For each genotype, at least 50 individual male crosses were
scored for eye color of female progeny lacking transposase. The
percentage of progeny from each repair class was calculated on a
per vial basis, with each vial representing a separate experiment.
Statistical comparisons were done with a Kruskal-Wallis non-
parametric ANOVA followed by Dunn’s multiple comparisons test
using InStat3 (GraphPad).
For analysis of HR synthesis tract lengths, genomic DNA was
purified and PCR reactions were performed as in [43], using
primer pairs with the internal primer located 250, 2420, and 4674
base pairs from the cut site at the 59 end of P{wa}.
For deletion analysis, the percentage of females with scalloped
wings was calculated relative to all yellow-eyed females counted.
The percentage of male lethal and small (0.1–3.6 kb) deletions was
calculated based on a subset of yellow-eyed females (one from each
original male parent) that were individually crossed to males
bearing the FM7w balancer. Vials for which no white-eyed male
progeny were recovered were scored as male lethal. Some of the
male lethal events also caused a scalloped-wing phenotype in
heterozygous females. For those that did not, testing to ensure that
the male lethality was due to deletion of scalloped coding sequence
was performed by recovering the repaired chromosomes in trans to
the hypomorphic sd1 mutation [79] and scoring for a scalloped-
wing phenotype. Repair events which could be recovered in males
were subjected to PCR analysis, using primers internal to P{wa}
[43], to detect small deletions into one or both introns of sd.
I-SceI break repair assayRepair of I-SceI mediated DNA double strand breaks was
studied in the context of the chromosomally integrated [Iw]7
construct [52], which contains a single target site for the I-SceI
endonuclease. DSBs were induced in the male pre-meiotic
germline by crossing females harboring [Iw]7 to males expressing
the I-SceI endonuclease from a second chromosomal location
under the control of either the hsp70 promoter (70[I-SceI]1A) [52]
or the ubiquitin promoter (UIE[I-SceI]2R) [50]. Independent
inaccurate end-joining repair events from the male pre-meiotic
germline were recovered in male progeny and DNA was isolated
for analysis [80]. PCR was performed using primers PE59
(GATAGCCGAAGCTTACCGAAGT) and jn39b (GGACATT-
GACGCTATCGACCTA) to amplify a 1.3 kb fragment of the
[Iw]7 construct including the I-SceI target site. Products were gel
purified (GenScript) and sequencing of PCR products was
performed using the PE59 primer. Sequences were aligned using
ClustalW or by manual inspection against sequence obtained from
an uncut [Iw]7 construct. Statistical comparisons were done using
Excel and SPSS.
Supporting Information
Figure S1 Polymerase theta orthologues from various metazo-
ans. The conserved helicase-like (blue oval) and polymerase (pink
box) domains are indicated. All of the orthologs have additional
conserved regions in the N and C-termini (white boxes), separated
by a variable-length linker region.
Found at: doi:10.1371/journal.pgen.1001005.s001 (0.51 MB PPT)
Figure S2 Sequence changes in different mus308 mutant alleles,
compared to the Drosophila reference genome sequence. Allele-
specific changes are highlighted in yellow. 1 +1 corresponds to the
‘A’ in the start codon of mus308.
Found at: doi:10.1371/journal.pgen.1001005.s002 (0.11 MB PPT)
Figure S3 Alignment of conserved N-terminus of mus308
orthologs. Dmel, Drosophila melanogaster; Agam, Anopheles gambia;
Mmus, Mus musculus; Hsap, Homo sapiens; Drer, Danio rerio; Atha,
Arabidopsis thaliana; Cele, Caenorhabditis elegans. Conserved amino
acids are indicated below each alignment. The red arrow
corresponds to the G621S substitution in the 3294 allele, the blue
arrow corresponds to the P781L substitution in the D5 allele.
Found at: doi:10.1371/journal.pgen.1001005.s003 (0.19 MB PPT)
Acknowledgments
We are grateful to members of the McVey, Mirkin, and Freudenreich labs
for discussions that informed this work and to Jeff Sekelsky and Graham
Walker for helpful comments. We would also like to thank the three
anonymous reviewers whose suggestions greatly improved the paper. We
thank the Bloomington stock center and Zuker labs for mus308 mutant
alleles, Bill Engels for the UIE-I-SceI stock, and Yikang Rong for the hsp70-
I-SceI and [Iw]7 stocks.
Author Contributions
Conceived and designed the experiments: SHC AMY MM. Performed the
experiments: SHC AMY. Analyzed the data: SHC AMY MM.
Contributed reagents/materials/analysis tools: SHC MM. Wrote the
paper: AMY MM.
References
1. Lehoczky P, McHugh PJ, Chovanec M (2007) DNA interstrand cross-link repair
in Saccharomyces cerevisiae. FEMS Microbiol Rev 31: 109–133.
2. Li X, Heyer WD (2008) Homologous recombination in DNA repair and DNA
damage tolerance. Cell Res 18: 99–113.
DNA Pol h Promotes Alt-EJ
PLoS Genetics | www.plosgenetics.org 14 July 2010 | Volume 6 | Issue 7 | e1001005
3. Pardo B, Gomez-Gonzalez B, Aguilera A (2009) DNA repair in mammalian
cells: DNA double-strand break repair: how to fix a broken relationship. Cell
Mol Life Sci 66: 1039–1056.
4. Nohmi T, Masumura K (2005) Molecular nature of intrachromosomal deletions
and base substitutions induced by environmental mutagens. Environ Mol
Mutagen 45: 150–161.
5. Raschle M, Knipscheer P, Enoiu M, Angelov T, Sun J, et al. (2008) Mechanism
of replication-coupled DNA interstrand crosslink repair. Cell 134: 969–980.
6. Weterings E, Chen DJ (2008) The endless tale of non-homologous end-joining.
Cell Res 18: 114–124.
7. Lieber MR, Lu H, Gu J, Schwarz K (2008) Flexibility in the order of action and
in the enzymology of the nuclease, polymerases, and ligase of vertebrate non-
homologous DNA end joining: relevance to cancer, aging, and the immune
system. Cell Res 18: 125–133.
8. McVey M, Lee SE (2008) MMEJ repair of double-strand breaks (director’s cut):
deleted sequences and alternative endings. Trends Genet 24: 529–538.
9. Simsek D, Jasin M (2010) Alternative end-joining is suppressed by the canonical
NHEJ component Xrcc4-ligase IV during chromosomal translocation forma-
tion. Nat Struct Mol Biol 17: 410–416.
10. Rass E, Grabarz A, Plo I, Gautier J, Bertrand P, et al. (2009) Role of Mre11 in
chromosomal nonhomologous end joining in mammalian cells. Nat Struct Mol
Biol 16: 819–824.
11. Ma JL, Kim EM, Haber JE, Lee SE (2003) Yeast Mre11 and Rad1 proteins
define a Ku-independent mechanism to repair double-strand breaks lacking
overlapping end sequences. Mol Cell Biol 23: 8820–8828.
12. Weinstock DM, Brunet E, Jasin M (2007) Formation of NHEJ-derived
reciprocal chromosomal translocations does not require Ku70. Nat Cell Biol
9: 978–981.
13. Zhu C, Mills KD, Ferguson DO, Lee C, Manis J, et al. (2002) Unrepaired DNA
breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to
translocations. Cell 109: 811–821.
14. Bentley J, Diggle CP, Harnden P, Knowles MA, Kiltie AE (2004) DNA double
strand break repair in human bladder cancer is error prone and involves
microhomology-associated end-joining. Nucleic Acids Res 32: 5249–5259.
15. Mattarucchi E, Guerini V, Rambaldi A, Campiotti L, Venco A, et al. (2008)
Microhomologies and interspersed repeat elements at genomic breakpoints in
chronic myeloid leukemia. Genes Chromosomes Cancer 47: 625–632.
16. Corneo B, Wendland RL, Deriano L, Cui X, Klein IA, et al. (2007) Rag
mutations reveal robust alternative end joining. Nature 449: 483–486.
17. McVey M, Radut D, Sekelsky JJ (2004) End-joining repair of double-strand
breaks in Drosophila melanogaster is largely DNA ligase IV independent.
Genetics 168: 2067–2076.
18. Sekelsky JJ, Brodsky MH, Burtis KC (2000) DNA repair in Drosophila: insights
from the Drosophila genome sequence. J Cell Biol 150: F31–36.
19. Takasu-Ishikawa E, Yoshihara M, Hotta Y (1992) Extra sequences found at P
element excision sites in Drosophila melanogaster. Mol Gen Genet 232: 17–23.
20. Ducau J, Bregliano JC, de La Roche Saint-Andre C (2000) Gamma-irradiation
stimulates homology-directed DNA double-strand break repair in Drosophila
embryo. Mutat Res 460: 69–80.
21. Welzel N, Le T, Marculescu R, Mitterbauer G, Chott A, et al. (2001) Templated
nucleotide addition and immunoglobulin JH-gene utilization in t(11;14)
junctions: implications for the mechanism of translocation and the origin of
mantle cell lymphoma. Cancer Res 61: 1629–1636.
22. Jager U, Bocskor S, Le T, Mitterbauer G, Bolz I, et al. (2000) Follicular
lymphomas’ BCL-2/IgH junctions contain templated nucleotide insertions:
novel insights into the mechanism of t(14;18) translocation. Blood 95:
3520–3529.
23. Denny CT, Hollis GF, Magrath IT, Kirsch IR (1985) Burkitt lymphoma cell line
carrying a variant translocation creates new DNA at the breakpoint and violates
the hierarchy of immunoglobulin gene rearrangement. Mol Cell Biol 5:
3199–3207.
24. Verkaik NS, Esveldt-van Lange RE, van Heemst D, Bruggenwirth HT,
Hoeijmakers JH, et al. (2002) Different types of V(D)J recombination and end-
joining defects in DNA double-strand break repair mutant mammalian cells.
Eur J Immunol 32: 701–709.
25. Lundberg R, Mavinakere M, Campbell C (2001) Deficient DNA end joining
activity in extracts from fanconi anemia fibroblasts. J Biol Chem 276:
9543–9549.
26. Boyd JB, Sakaguchi K, Harris PV (1990) mus308 mutants of Drosophila exhibit
hypersensitivity to DNA cross-linking agents and are defective in a deoxyribo-
nuclease. Genetics 125: 813–819.
27. Harris PV, Mazina OM, Leonhardt EA, Case RB, Boyd JB, et al. (1996)
Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair
with homology to prokaryotic DNA polymerase I genes. MCB 16: 5764–5771.
28. Muzzini DM, Plevani P, Boulton SJ, Cassata G, Marini F (2008) Caenorhabditis
elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-
link repair pathways. DNA Repair (Amst) 7: 941–950.
29. Inagaki S, Suzuki T, Ohto MA, Urawa H, Horiuchi T, et al. (2006) Arabidopsis
TEBICHI, with helicase and DNA polymerase domains, is required for
regulated cell division and differentiation in meristems. Plant Cell 18: 879–892.
30. Seki M, Marini F, Wood RD (2003) POLQ (Pol theta), a DNA polymerase and
DNA-dependent ATPase in human cells. Nucleic Acids Res 31: 6117–6126.
31. Shima N, Munroe RJ, Schimenti JC (2004) The mouse genomic instability
mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm.
Mol Cell Biol 24: 10381–10389.
32. Zan H, Shima N, Xu Z, Al-Qahtani A, Evinger Iii AJ, et al. (2005) The
translesion DNA polymerase theta plays a dominant role in immunoglobulin
gene somatic hypermutation. Embo J 24: 3757–3769.
33. Masuda K, Ouchida R, Hikida M, Nakayama M, Ohara O, et al. (2006)
Absence of DNA polymerase theta results in decreased somatic hypermutation
frequency and altered mutation patterns in Ig genes. DNA Repair (Amst) 5:
1384–1391.
34. Seki M, Masutani C, Yang LW, Schuffert A, Iwai S, et al. (2004) High-efficiency
bypass of DNA damage by human DNA polymerase Q. Embo J 23: 4484–4494.
35. Yoshimura M, Kohzaki M, Nakamura J, Asagoshi K, Sonoda E, et al. (2006)
Vertebrate POLQ and POLbeta cooperate in base excision repair of oxidative
DNA damage. Mol Cell 24: 115–125.
36. Prasad R, Longley MJ, Sharief FS, Hou EW, Copeland WC, et al. (2009)
Human DNA polymerase {theta} possesses 59-dRP lyase activity and functions
in single-nucleotide base excision repair in vitro. Nucleic Acids Res 37:
1868–1877.
37. Kawamura K, Bahar R, Seimiya M, Chiyo M, Wada A, et al. (2004) DNA
polymerase theta is preferentially expressed in lymphoid tissues and upregulated
in human cancers. Int J Cancer 109: 9–16.
38. Pang M, McConnell M, Fisher PA (2005) The Drosophila mus 308 gene
product, implicated in tolerance of DNA interstrand crosslinks, is a nuclear
protein found in both ovaries and embryos. DNA Repair (Amst) 4: 971–982.
39. Arana ME, Seki M, Wood RD, Rogozin IB, Kunkel TA (2008) Low-fidelity
DNA synthesis by human DNA polymerase theta. Nucleic Acids Res 36:
3847–3856.
40. Boyd JB, Golino MD, Shaw KES, Osgood CJ, Green MM (1981) Third-
chromosome mutagen-sensitive mutants of Drosophila melanogaster. Genetics 97:
607–623.
41. Laurencon A, Orme CM, Peters HK, Boulton CL, Vladar EK, et al. (2004) A
large-scale screen for mutagen-sensitive loci in Drosophila. Genetics 167:
217–231.
42. Staeva-Vieira E, Yoo S, Lehmann R (2003) An essential role of DmRad51/
SpnA in DNA repair and meiotic checkpoint control. Embo J 22: 5863–5874.
43. Adams MD, McVey M, Sekelsky JJ (2003) Drosophila BLM in double-strand
break repair by synthesis-dependent strand annealing. Science 299: 265–267.
44. Beall EL, Rio DC (1997) Drosophila P-element transposase is a novel site-
specific endonuclease. Genes Dev 11: 2137–2151.
45. McVey M, Adams M, Staeva-Vieira E, Sekelsky JJ (2004) Evidence for multiple
cycles of strand invasion during repair of double-strand gaps in Drosophila.
Genetics 167: 699–705.
46. Johnson-Schlitz DM, Flores C, Engels WR (2007) Multiple-pathway analysis of
double-strand break repair mutations in Drosophila. PLoS Genet 3: e50.
doi:10.1371/journal.pgen.0030050.
47. Wei DS, Rong YS (2007) A genetic screen for DNA double-strand break repair
mutations in Drosophila. Genetics 177: 63–77.
48. McVey M, Larocque JR, Adams MD, Sekelsky JJ (2004) Formation of deletions
during double-strand break repair in Drosophila DmBlm mutants occurs after
strand invasion. Proc Natl Acad Sci U S A 101: 15694–15699.
49. Srivastava A, Simmonds AJ, Garg A, Fossheim L, Campbell SD, et al. (2004)
Molecular and functional analysis of scalloped recessive lethal alleles in
Drosophila melanogaster. Genetics 166: 1833–1843.
50. Preston CR, Flores CC, Engels WR (2006) Differential usage of alternative
pathways of double-strand break repair in Drosophila. Genetics 172:
1055–1068.
51. Weinstock DM, Nakanishi K, Helgadottir HR, Jasin M (2006) Assaying double-
strand break repair pathway choice in mammalian cells using a targeted
endonuclease or the RAG recombinase. Methods Enzymol 409: 524–540.
52. Rong YS, Golic KG (2003) The homologous chromosome is an effective
template for the repair of mitotic DNA double-strand breaks in Drosophila.
Genetics 165: 1831–1842.
53. Yu AM, McVey M (2010) Synthesis-dependent microhomology-mediated end
joining accounts for multiple types of repair junctions. Nucleic Acids Res. 2010
May 11. [Epub ahead of print].
54. Lee K, Lee SE (2007) Saccharomyces cerevisiae Sae2- and Tel1-dependent
single-strand DNA formation at DNA break promotes microhomology-mediated
end joining. Genetics 176: 2003–2014.
55. Decottignies A (2007) Microhomology-mediated end joining in fission yeast is
repressed by pku70 and relies on genes involved in homologous recombination.
Genetics 176: 1403–1415.
56. Taylor EM, Cecillon SM, Bonis A, Chapman JR, Povirk LF, et al. (2010) The
Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in
Xenopus laevis. Nucleic Acids Res 38: 441–454.
57. Dinkelmann M, Spehalski E, Stoneham T, Buis J, Wu Y, et al. (2009) Multiple
functions of MRN in end-joining pathways during isotype class switching. Nat
Struct Mol Biol 16: 808–813.
58. Xie A, Kwok A, Scully R (2009) Role of mammalian Mre11 in classical and
alternative nonhomologous end joining. Nat Struct Mol Biol 16: 814–818.
59. Deriano L, Stracker TH, Baker A, Petrini JH, Roth DB (2009) Roles for NBS1
in alternative nonhomologous end-joining of V(D)J recombination intermedi-
ates. Mol Cell 34: 13–25.
DNA Pol h Promotes Alt-EJ
PLoS Genetics | www.plosgenetics.org 15 July 2010 | Volume 6 | Issue 7 | e1001005
60. Nassif N, Penney J, Pal S, Engels WR, Gloor GB (1994) Efficient copying of
nonhomologous sequences from ectopic sites via P-element-induced gap repair.
Mol Cell Biol 14: 1613–1625.
61. Yusufzai T, Kadonaga JT (2008) HARP is an ATP-driven annealing helicase.
Science 322: 748–750.
62. Brissett NC, Pitcher RS, Juarez R, Picher AJ, Green AJ, et al. (2007) Structure of
a NHEJ polymerase-mediated DNA synaptic complex. Science 318: 456–459.
63. Audebert M, Salles B, Calsou P (2004) Involvement of poly(ADP-ribose)
polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA
double-strand breaks rejoining. J Biol Chem 279: 55117–55126.
64. Wang H, Rosidi B, Perrault R, Wang M, Zhang L, et al. (2005) DNA ligase III
as a candidate component of backup pathways of nonhomologous end joining.
Cancer Res 65: 4020–4030.
65. Grossmann KF, Ward AM, Matkovic ME, Folias AE, Moses RE (2001) S.
cerevisiae has three pathways for DNA interstrand crosslink repair. Mutat Res
487: 73–83.
66. Sarkar S, Davies AA, Ulrich HD, McHugh PJ (2006) DNA interstrand crosslink
repair during G1 involves nucleotide excision repair and DNA polymerase zeta.
Embo J 25: 1285–1294.
67. Jonnalagadda VS, Matsuguchi T, Engelward BP (2005) Interstrand crosslink-
induced homologous recombination carries an increased risk of deletions and
insertions. DNA Repair (Amst) 4: 594–605.
68. Liu Y, Nairn RS, Vasquez KM (2008) Processing of triplex-directed psoralen
DNA interstrand crosslinks by recombination mechanisms. Nucleic Acids Res
36: 4680–4688.
69. Zheng H, Wang X, Legerski RJ, Glazer PM, Li L (2006) Repair of DNA
interstrand cross-links: interactions between homology-dependent and homol-
ogy-independent pathways. DNA Repair (Amst) 5: 566–574.
70. Scaringe WA, Li K, Gu D, Gonzalez KD, Chen Z, et al. (2008) Somatic
microindels in human cancer: the insertions are highly error-prone and derive
from nearby but not adjacent sense and antisense templates. Hum Mol Genet
17: 2910–2918.71. Kusano K, Johnson-Schlitz DM, Engels WR (2001) Sterility of Drosophila with
mutations in the Bloom syndrome gene–complementation by Ku70. Science
291: 2600–2602.72. Windhofer F, Krause S, Hader C, Schulz WA, Florl AR (2008) Distinctive
differences in DNA double-strand break repair between normal urothelial andurothelial carcinoma cells. Mutat Res 638: 56–65.
73. Marculescu R, Vanura K, Montpellier B, Roulland S, Le T, et al. (2006)
Recombinase, chromosomal translocations and lymphoid neoplasia: Targetingmistakes and repair failures. DNA Repair (Amst) 5: 1246–1258.
74. Okamura K, Chung WJ, Ruby JG, Guo H, Bartel DP, et al. (2008) TheDrosophila hairpin RNA pathway generates endogenous short interfering
RNAs. Nature 453: 803–806.75. Czech B, Malone CD, Zhou R, Stark A, Schlingeheyde C, et al. (2008) An
endogenous small interfering RNA pathway in Drosophila. Nature 453:
798–802.76. Koundakjian EJ, Cowan DM, Hardy RW, Becker AH (2004) The Zuker
Collection: A Resource for the Analysis of Autosomal Gene Function inDrosophila melanogaster. Genetics 167: 203–206.
77. McVey M, Andersen SL, Broze Y, Sekelsky J (2007) Multiple functions of
Drosophila BLM helicase in maintenance of genome stability. Genetics 176:1979–1992.
78. Kurkulos M, Weinberg JM, Roy D, Mount SM (1994) P element-mediated in
vivo deletion analysis of white-apricot: deletions between direct repeats are strongly
favored. Genetics 136: 1001–1011.79. Zider A, Paumard-Rigal S, Frouin I, Silber J (1998) The vestigial gene of
Drosophila melanogaster is involved in the formation of the peripheral nervous
system: genetic interactions with the scute gene. J Neurogenet 12: 87–99.80. Gloor GB, Preston CR, Johnson-Schlitz DM, Nassif NA, Phillis RW, et al.
(1993) Type I repressors of P element mobility. Genetics 135: 81–95.
DNA Pol h Promotes Alt-EJ
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